This research will use a molecular genetic approach to examine the regulation of a major intersection between catabolic and anabolic pathways in eucaryotic cells. Our focus is the family of isozymes of isocitrate dehydrogenase and our goal is to determine the role of these enzymes in controlling metabolic flux of carbon and reducing equivalents across compartmental barriers. These isozymes include a mitochondrial NAD+- specific enzyme (IDH), which catalyzes an allosterically regulated reaction in the tricarboxylic acid cycle, and two NADP+-specific enzymes, which are structurally similar but differentially localized in mitochondrial (IDP1) and cytosolic (IDP2) cellular compartments. Previous efforts have been focused on cloning and sequence analysis of the genes from Saccharomyces cerevisiae encoding each isozyme. Initial experiments in this proposal will examine the growth phenotypes and metabolic defects associated with loss of each isozyme by construction of mutant yeast strains containing all possible combinations of disruptions in genes encoding the isocitrate dehydrogenases. Subsequent experiments will test the importance of allosteric regulation and of mitochondrial localization for function of the NAD+-specific enzyme in the tricarboxylic acid cycle in vivo. This will be achieved by expression of mutant forms of that enzyme in yeast strains with defined metabolic defects. In other experiments, a collection of yeast mutants lacking combinations of the NADP+-specific isozymes and of a functional hexose monophosphate pathway will be constructed to assess relative contributions to cellular pools of biosynthetic NADPH reducing equivalents. Finally, these studies will be extended to mammalian systems with analyses of sequences and expression of cDNAs for the isocitrate dehydrogenases.